Duang S, Zhang M, Liu C, Dong Q
Department of Nephrology, Affiliated Hospital of Chengde Medical University, Chengde, China.
J Physiol Pharmacol. 2022 Oct;73(5). doi: 10.26402/jpp.2022.5.03. Epub 2023 Mar 16.
Vascular smooth muscle cells (VSMCs) play a key role in vascular calcification, which is associated with enhanced mortality in chronic kidney disease. To ascertain the concentration of parathyroid hormone (PTH) that induces apoptosis of VSMCs and to explore whether the mechanism of endoplasmic reticulum (ER) stress is involved in PTH-induced calcification of VSMCs. The appropriate concentration and intervention time of PTH-inducing apoptosis of VSMCs were screened by flow cytometry. To investigate the effects of PTH on ER stress-related and apoptotic proteins in VSMCs, they were divided into four groups. These were the control group, the PTH group, the siC/EBP homologous protein (CHOP) + PTH group (in which siCHOP was used to knockdown the expression of CHOP at first), and the sp600125 + PTH group (in which the cells were pretreated with sp600125 at a concentration of 10 ng/ml for 24 h at first). Then, all groups except the control group were given 1x10 mol/L PTH to stimulate VSMCs. The changes in ER stress and apoptosis-related proteins in each group were detected, and the cell calcification was tested by Alizarin Red staining. Flow cytometry showed that the concentration of 1x10 mol/L PTH induced apoptosis most significantly. The apoptosis rate of the cells increased with the extension of stimulation time. The apoptosis of VSMCs pretreated with the Jun-N-terminal kinase (JNK) antagonist sp600125 was significantly reduced. The expression of cleaved caspase-3, PERK, IRE1, and CHOP was detected by Western blot analysis when cells were stimulated with 10 mol/L PTH for 14 days. After the knockdown of the CHOP expression by siCHOP, cleaved caspase-3 expression was significantly reduced. Alizarin Red staining showed siCHOP and sp600125 inhibits the VSMCs' calcification induced by PTH. In conclusion: the ER stress mechanism is involved in VSMCs' calcification induced by PTH.
血管平滑肌细胞（VSMCs）在血管钙化中起关键作用，而血管钙化与慢性肾脏病死亡率升高相关。为确定诱导VSMCs凋亡的甲状旁腺激素（PTH）浓度，并探究内质网（ER）应激机制是否参与PTH诱导的VSMCs钙化。通过流式细胞术筛选PTH诱导VSMCs凋亡的合适浓度和干预时间。为研究PTH对VSMCs中ER应激相关蛋白和凋亡蛋白的影响，将其分为四组。分别为对照组、PTH组、siC/EBP同源蛋白（CHOP）+PTH组（先用siCHOP敲低CHOP表达）和sp600125+PTH组（先用浓度为10 ng/ml的sp600125预处理细胞24小时）。然后，除对照组外，所有组均给予1×10⁻⁷mol/L PTH刺激VSMCs。检测每组中ER应激和凋亡相关蛋白的变化，并用茜素红染色检测细胞钙化情况。流式细胞术显示1×10⁻⁷mol/L PTH诱导凋亡最显著。细胞凋亡率随刺激时间延长而增加。用Jun氨基末端激酶（JNK）拮抗剂sp600125预处理的VSMCs凋亡明显减少。当细胞用10⁻⁷mol/L PTH刺激14天时，通过蛋白质免疫印迹分析检测裂解的半胱天冬酶-3、PERK、IRE1和CHOP的表达。用siCHOP敲低CHOP表达后，裂解的半胱天冬酶-3表达明显降低。茜素红染色显示siCHOP和sp600125抑制PTH诱导的VSMCs钙化。总之：ER应激机制参与PTH诱导的VSMCs钙化。
